Nucleic acids are amplified and act as catalysts and ligands through complementarity of A-T(U) and G-C base pairs. However, unlike 20 different amino acids in natural proteins, natural nucleic acids are composed of nucleotides consisting of only 4 different bases. This number limit restricts the functions of DNA and RNA molecules. Unnatural base pair systems provide a resolution to this problem because they increase the types of nucleic acid bases to allow expansion of genetic information (Non-patent Documents 1-5). Unnatural base pairs are required to have highly specific complementarity which allows site-specific incorporation of special nucleotide analogs into DNA and RNA through polymerase-catalyzed reactions. If this requirement is achieved, current genetic engineering technology, which is limited by the number of naturally-occurring bases, can be replaced with a novel technology using unnatural base pair systems.
The first attempt to create unnatural base pairs was made by Benner et al (Non-patent Documents 6-7). They developed some unnatural base pairs, including isoguanine-isocytosine (isoG-isoC) and xanthosine-diaminopyrimidine, based on different hydrogen bonding patterns than those of natural base pairs. Recently, these unnatural base pairs have been applied to PCR amplification (Non-patent Documents 8-9) and sequence analysis (Non-patent Document 10) of DNA fragments containing these base pairs. However, the fidelity is relatively not high and/or complicated procedures are required. In addition to these problems, 2-aminopyrimidine analogs such as isoC and diaminopyrimidine are not recognized as substrates by T7 RNA polymerase. Thus, these base pairs are of limited use.
Subsequently, Kool et al. synthesized hydrophobic bases having shapes similar to those of natural bases, but lacking the ability to form a hydrogen bond during base pairing (Non-patent Documents 11-12). These hydrophobic bases were selectively recognized by DNA polymerases, suggesting that geometric shape complementarity between paring bases is more important during replication, rather than hydrogen bonding interaction. Recently, a series of hydrophobic base pairs have been developed by Romesberg et al. and introduced into DNA in a complementary manner by the action of the Klenow fragment of E. coli-derived DNA polymerase I (Non-patent Documents 13-15). However, these hydrophobic bases did not conform to shape complementarity during replication, and their non-selective introduction through enzymatic reactions occurred between hydrophobic bases (Non-patent Document 14). Moreover, there is no report of these hydrophobic base pairs functioning during transcription.
By combining the ideas of hydrogen bonding pattern and shape complementarity, the inventors of the present invention developed unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxopyridine (y) (Non-patent Documents 16-17) as well as between 2-amino-6-(2-thiazolyl)purine (v) and y (Non-patent Document 18). The bulky substituents at the 6-position of s and v efficiently prevented undesirable base pairing (non-cognate pairing) with a natural base, and a substrate (nucleoside 5′-triphosphate) of y or modified y was introduced in a site-specific manner into RNA opposite s or v in the template by the action of T7 RNA polymerase. This specific transcription is available for practical use as a means for developing functional RNA molecules (Non-patent Documents 19-21), but the selectivity of s-y and v-y base pairings during replication is not notably higher than that during transcription (Non-patent Documents 16 and 18).
To solve the problems stated above, there is a demand for a novel artificial base pair showing excellent efficiency and selectivity during replication and transcription (for design of functional nucleic acids) or during all of replication, transcription and translation (for design of functional proteins).
The following documents are listed as reference documents, the entire contents of which are incorporated herein by reference.                Patent Document 1: WO2001/005801        Patent Document 2: WO2004/007713        Patent Document 3: WO2005/026187        Patent Document 4: Japanese Patent Application No. 2005-226492        
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